The present invention is directed to peptide nucleic acids and to methods of inhibiting bacterial growth and/or bacterial gene expression using peptide nucleic acids.
From the discovery of penicillin by Fleming in 1940""s there has been a constant search for new antibiotics, which search continues to this day. Although many antibiotics have been discovered, there is an on-going need for the discovery of new antibiotic compounds because of the emergence of drug resistant strains of bacteria. Thus, research on bacterial infection is a perpetual cycle of development of new antibiotics. When penicillin was first discovered, its broad-spectrum antibiotic activity was hailed as the xe2x80x9cmagic bulletxe2x80x9d in fighting many bacterial infections. However, over the years, many strains of bacteria have developed resistance to penicillin and other currently available antibiotic drugs. No antibiotic drug is effective against all bacterial infections. Many antibiotic drugs available today have a narrow spectrum of activity. That is, they are effective against only few specific types of bacterial infections. Thus, for example, the majority of current antibiotic drugs are ineffective against syphilis. and tuberculosis. In addition, some strains of syphilis, tuberculosis and other bacteria have developed resistance to currently available antibiotic drugs, which were effective drugs in the past.
Oligonucleotides and their analogs have been developed and used in molecular biology in certain procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with non isotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. These modifications include use of methyl phosphonates, phosphorothioates, phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Further modifications include modifications made to modulate uptake and cellular distribution. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for various disease states including use as antiviral agents. With the success of these oligonucleotides for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotide analogs.
Oligonucleotides can interact with native DNA and RNA in several ways. One of these is duplex formation between an oligonucleotide and a single stranded nucleic acid. The other is triplex formation between an oligonucleotide and double stranded DNA to form a triplex structure.
Peptide nucleic acids are compounds that in certain respects are similar to oligonucleotide analogs however in other very important respects their structure is very different. In peptide nucleic acids, the deoxyribose backbone of oligonucleotides has been replaced with a backbone more akin to a peptide than a sugar. Each subunit, or monomer, has a naturally occurring or non naturally occurring base attached to this backbone. One such backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds. Because of the radical deviation from the deoxyribose backbone, these compounds were named peptide nucleic acids (PNAs).
PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes as determined by Tm""s. This high thermal stability might be attributed to the lack of charge repulsion due to the neutral backbone in PNA. The neutral backbone of the PNA also results in the Tm""s of PNA/DNA(RNA) duplex being practically independent of the salt concentration. Thus the PNA/DNA duplex interaction offers a further advantage over DNA/DNA duplex interactions which are highly dependent on ionic strength. Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)2/DNA(RNA) triplexes of high thermal stability (see, e.g., Egholm, et al., Science, 1991, 254, 1497; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 9677).
In addition to increased affinity, PNA has also been shown to bind to DNA with increased specificity. When a PNA/DNA duplex mismatch is melted relative to the DNA/DNA duplex there is seen an 8 to 20xc2x0 C. drop in the Tm. This magnitude of a drop in Tm is not seen with the corresponding DNA/DNA duplex with a mismatch present.
The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations. The orientation is said to be anti-parallel when the DNA or RNA strand in a 5xe2x80x2 to 3xe2x80x2 orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5xe2x80x2 end of the DNA or RNA and amino end of the PNA is directed towards the 3xe2x80x2 end of the DNA or RNA. In the parallel orientation the carboxyl end and amino end of the PNA are just the reverse with respect to the 5xe2x80x2-3xe2x80x2 direction of the DNA or RNA.
PNAs bind to both single stranded DNA and double stranded DNA. As noted above, in binding to double stranded DNA it has been observed that two strands of PNA can bind to the DNA. While PHA/DNA duplexes are stable in the antiparallel configuration, it was previously believed that the parallel orientation is preferred for (PNA)2/DNA triplexes.
The binding of two single stranded pyrimidine PNAs to a double stranded DNA has been shown to take place via strand displacement, rather than conventional triple helix formation as observed with triplexing oligonucleotides. When PNAs strand invade double stranded DNA, one strand of the DNA is displaced and forms a loop on the side of the PNA2/DNA complex area. The other strand of the DNA is locked up in the (PNA)2/DNA triplex structure. The loop area (alternately referenced as a D loop) being single stranded, is susceptible to cleavage by enzymes that can cleave single stranded DNA.
A further advantage of PNA compared to oligonucleotides is that their polyamide backbones (having appropriate nucleobases or other side chain groups attached thereto) is not recognized by either nucleases or proteases and are not cleaved. As a result PNAs are resistant to degradation by enzymes unlike nucleic acids and peptides.
Because of their properties, PNAs are known to be useful in a number of different areas. Since PNAs have stronger binding and greater specificity than oligonucleotides, they are used as probes in cloning, blotting procedures, and in applications such as fluorescence in situ hybridization (FISH). Homopyrimidine PNAs are used for strand displacement in homopurine targets. The restriction sites that overlap with or are adjacent to the P-loop will not be cleaved by restriction enzymes. Also, the local triplex inhibits gene transcription. Thus in binding of PNAs to specific restriction sites within a DNA fragment, cleavage at those sites can be inhibited. Advantage can be taken of this in cloning and subcloning procedures. Labeled PNAs are also used to directly map DNA molecules. In effecting this, PNA molecules having a fluorescent label are hybridized to complementary sequences in duplex DNA using strand invasion.
PNAs have further been used to detect point mutations in PCR-based assays (PCR clamping). PCR clamping uses PNA to detect point mutations in a PCR-based assay, e.g. the distinction between a common wild type allele and a mutant allele, in a segment of DNA under investigation. A PNA oligomer complementary to the wild type sequence is synthesized. The PCR reaction mixture contains this PNA and two DNA primers, one of which is complementary to the mutant sequence. The wild type PNA oligomer and the DNA primer compete for hybridization to the target. Hybridization of the DNA primer and subsequent amplification will only occur if the target is a mutant allele. With this method, one can determine the presence and exact identity of a mutant.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs that bind complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics. PCT/EP/01219 describes novel peptide nucleic acid (PHA) compounds which bind complementary DNA and RNA more tightly than the corresponding DNA. Because of these binding properties as well as their stability, such PNA compounds find many uses in diagnostics and research reagents uses associated with both DNA and RNA. With complementary DNA and RNA they can form double-stranded, helical structures mimicking doublestranded DNA, and they are capable of being derivatized to bear pendant groups to further enhance or modulate their binding, cellular uptake, or other activity.
Specific sequence recognition of DNA or RNA is of increasing importance for the development of biological probes and new reagents for use in research (Uhlmann, E., Peyman, A., Chem. Rev., 1990, 90, 544, and Helene, C., Toulme, J. J., Biochim. Biophys. Acta., 1990, 1049, 99). Peptide nucleic acid (PNA), have properties making them well suited for use as biological probes and other applications. PNA have shown strong binding affinity and specificity to complementary DNA, sequence specific inhibition of DNA restriction enzyme cleavage and site specific in vitro inhibition of translation (Egholm, M., et al., Chem. Soc., Chem. Commun., 1993, 800; Egholm, M., et.al., Nature, 1993, 365, 566; and Nielsen, P., et.al. Nucl. Acids Res., 1993, 21, 197). Furthermore, PNA""s show nuclease resistance and stability in cell-extracts (Demidov, V. V., et al., Biochem. Pharmacol., 1994, 48, 1309-1313). Modifications of PNA include extended backbones (Hyrup, B., et.al. Chem. Soc., Chem. Commun., 1993, 518), extended linkers between the backbone and the nucleobase, reversal of the amido bond (Lagriffoul, P. H., et.al., Biomed. Chem. Lett., 1994, 4, 1081), and the use of a chiral backbone based on alanine (Dueholm, K. L, et.al., BioMed. Chem. Lett., 1994, 4, 1077).
A method of inhibiting protein synthesis by contacting 28S rRNA of a protein synthesizing system with a protein synthesis inhibitory amount of an oligonucleotide that hybridizes to the sarcin recognition domain loop of the 28S rRNA has been previously reported (U.S. Pat. No. 5,220,014, entitled rRNA Specific Oligonucleotides, issued Jun. 15, 1993).
Antibacterial activity and inhibition of protein synthesis in E. coli has been reported using DNA analogs having methylcarbonate internucleoside linkages in place of phosphodiester linkages (Rahman, M. A., et al., Antisense Research and Development, 1991, 1, 319-327).
The 3xe2x80x2 end of the 16S rRNA of E. coli has been targeted by a complementary pentanucleotide. The initiation of protein biosynthesis is thereby blocked (Eckhardt, H., Luhrmann, R., J. Biol. Chem., .1979, 254, 11185-11188).
Selective inhibition of E. coli protein synthesis and growth by nonionic oligonucleotides (methylphosphonate linkages) complementary to the 3xe2x80x2 end of the 16S rRNA has been previously reported (Jayaraman, K., et al., Proc. Natl. Acad. Sci., 1981, 78, 1537-1541).
Oligodeoxyribonucleotides complementary to the 3xe2x80x2 terminal segment of the 16s-rRNA in molecules have shown suppression of translation in their ribosomes in an in-vitro assay (Korobkova, E. S., et al., Mikrobiol. Z., 1995, 57, 30-36).
Peptide Nucleic Acids are described in U.S. Pat. No. 5,539,082, issued Jul. 23, 1996, entitled Novel Peptide Nucleic Acids and U.S. Pat. No. 5,539,083, issued Jul. 23, 1996, entitled PNA Combinatorial Libraries and Improved Methods of Synthesis, the contents of which are hereby incorporated by reference. Peptide Nucleic Acids are further described in U.S. patent application Ser. No. 08/686,113, filed Jul. 24, 1996, entitled Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity, in which a supplemental notice of allowability dated Jun. 2, 1997, has been received, the contents of which is hereby incorporated by reference.
One aspect of the present invention provides methods of killing or inhibiting growth of a bacteria comprising contacting the bacteria with a peptide nucleic acid. The methods include employment of a peptide nucleic acid that is complementary to a region of the bacteria ribosomal RNA. The methods further include use of a peptide nucleic acid that is complementary to a region of the bacteria messenger RNA. In one aspect of the invention, the methods include contacting the bacteria with at least one PNA or PNA-linked antibiotic. In one embodiment of the invention, peptide nucleic acids are from 5 to about 40 monomer units in length. In a preferred embodiment of the invention peptide nucleic acids are from about 5 to about 25 monomer units in length.
In a further aspect of the invention, the methods include peptide nucleic acid complementary to a region of the bacteria ribosomal RNA and a further peptide nucleic acid complementary to a region of the bacteria messenger RNA. The use of peptide nucleic acid to both ribosomal RNA and messenger RNA may further include at least one antibiotic.
A further aspect of the present invention provides an antibacterial composition comprising a peptide nucleic acid. In one embodiment the composition includes a further peptide nucleic acid. In a preferred embodiment the peptide nucleic acid has bacteriostatic or bacteriocidal properties. In one embodiment the peptide nucleic acid is targeted to an essential bacterial gene.
In another aspect, the present invention provides antibacterial pharmaceutical compositions comprising peptide nucleic acid. In one embodiment the peptide nucleic acid is targeted to an essential bacterial gene. In a preferred embodiment the targeted gene is encodes xcex2-lactamase. In a more preferred embodiment the antibacterial pharmaceutical composition further comprises a xcex2-lactam antibacterial agent.
The present invention further provides methods of treating a subject suffering from a bacterial infection by administration of a peptide nucleic acid. In one embodiment the method includes a peptide nucleic acid complementary to a region of the bacteria ribosomal RNA. In another embodiment the method includes a peptide nucleic acid complementary to a region of the bacteria mRNA. A further embodiment includes concurrent treatment with an antibiotic.
The present invention includes methods of disinfection comprising selecting an object to be disinfected and contacting the object with peptide nucleic acid. The peptide nucleic is removed by rinsing the object with a sterile liquid to remove the peptide nucleic acid. In a preferred embodiment, the peptide nucleic acid is in a solution and the object is contacted with the solution over all solvent accessible areas of the object.
The present invention relates to methods of killing or inhibiting the growth of bacteria through treatment with either PNAs alone or PNAS in combination with one or more antibiotics. rRNA targeted PNAs alone have bactericidal or growth inhibitory effects on selected bacteria. mRNA targeted PRAs alone are effective in selectively blocking production of selected proteins. mRNA targeted PNAs can be used to enhance the effects of selected antibiotics by targeting genes responsible for the production of proteins that inhibit the activity of the antibiotic. In a multi-drug approach, rRNA targeted PNAs, mRNA targeted PNAs and selected antibiotics can be used simultaneously to give a synergistic effect.
In one aspect of the present invention rRNA of E. coli is targeted by selected PNAs. rRNA is essential for the translation of mRNA in the production of proteins and has been reported to be a preferred target of inhibition of translation (Noller, H. F., et al., Science, 1992, 256, 1416-1419). There are a number of natural antibiotics that appear to act by binding to rRNA (Cundliff, E., (1989), In the Ribosome, Hill, et al., Eds., Am. Soc. Microbiol., Washington, D.C., 479-490).
The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations. The orientation is said to be anti-parallel when the DNA or RNA strand in a 5xe2x80x2 to 3xe2x80x2 orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5xe2x80x2 end of the DNA or RNA and amino end of the PNA is directed towards the 3xe2x80x2 end of the DNA or RNA. In the parallel orientation the carboxyl end and amino end of the PNA are just the reverse with respect to the 5xe2x80x2-3xe2x80x2 direction of the DNA or RNA.
In accordance with this invention, it has now been found that very stable triplexes are formed between two single stranded PNAs or a linked PNA (bis PNA) and a mRNA or rRNA target strand where the Watson/Crick base pairing strand is in an anti-parallel orientation relative to the target strand and the Hoogsteen base pairing strand is in a parallel orientation relative to the target strand. As so orientated to the target strand, the two PNA strands are therefore anti-parallel to each other. Such stability is very desirable.
In the Hoogsteen strand of the linked PNAs the cytosines have been replaced with pseudo isocytosines. Normal Hoogsteen binding requires that the cytosines be protonated. This makes the Hoogsteen strand binding pH dependent. We have previously found that replacement of one or more of the cytosine nucleobases in a Hoogsteen strand with pseudo isocytosine and other like nucleobases removes this dependence. The replacement of cytosine by pseudo isocytosine or other like C-pyrimidine nucleobases is effected in a straight forward manner as illustrated in the examples below.
Duplex and triplex forming PNAs were synthesized to anneal to the RNA component of the E. coli ribosome. The selected target regions of the rRNA was within the peptidyl transferase center, the xcex1-sarcin loop and the mRNA binding domain at the 3xe2x80x2 end of the 16S rRNA. These sites are functionally active and appear to be accessible for interactions with antibiotics, translation factors, structure probing agents, other RNA molecules and short oligonucleotides (Steitz, J. A., Jakes, K., PNAS, 1975, 72, 4634-4738; Engberg, J., et al., (1989) , In the Ribosome, Hill, et al., Eds., Am. Soc. Microbiol., Washington, D.C., 168-179; Hill, W. E., (1989), In the Ribosome, Hill, et al., Eds., Am. Soc. Microbiol., Washington, D.C., 253-261). The inhibition of rRNA in vitro by DNA oligonucleotides has been shown in previous studies (Taniguchi, T., Weissmann, C., Nature, 1978, 275, 770-772; Walker, K., et al., J. Biol. Chem., 1989, 265, 2428-2430; Saxena, S. K., Ackerman, E. J., J. Boil. Chem., 19909, 265, 3263-3269; Brigotti, M., et al., Biochem. Mol. Biol. Int., 1993, 31, 897-903; Meyer, H. A., et al., Nucl. Acids Res., 1996, 24, 3996-4002). It has also been shown that PNA targeted to template RNA within telomerase can inhibit its enzymatic activity (Norton, J. C., et al., Nature Biotechnology, 1996, 14, 615-619). Superior hybridization and stability of PNAs have been previously demonstrated via the blocking of polymerase and ribosome progression when bound to template sequences (Hanvey, J. C., et al., science, 1992, 258, 1481-1485; Nielsen, P. E., et al., gene, 1994, 149, 139-145; Knudsen, H., Nielsen, P. E., Nucl. Acids Res., 1996, 24, 494-500).
While not wanting to be bound by theory, it is believed that the PNAs targeted to rRNA effect killing or inhibition of bacterial growth by effecting gene expression at the level of translation. Supportive of this reasoning are the examples below. In identical in-vitro assays the inhibition of protein synthesis is determined first by colorimetric measurement of protein synthesis and second by measurement of radioactive methionine and UTP incorporated into protein and mRNA respectively. After the inhibition of xcex2-galactosidase synthesis was determined colorimetrically (Example 5) the assay was rerun using 32P-UTP and 35S-methionine (Example 6). The level of radioactive methionine incorporation decreased while the level of radioactive UTP remained constant with increasing amounts of rRNA targeted PNA added to the assay. The data show uninterrupted transcription with a steady decrease in translation.
In another aspect of the present invention, methods of killing or inhibiting the growth of a bacteria are effected through the use of PNAs targeted to mRNA. The advantage to using mRNA targeted PNAs is that only specific gene products need be targeted, mRNA targeted PNAs can be targeted to core proteins necessary for the survival and replication of bacterial cells thereby inhibiting cell growth. PNAs can also be targeted to specific mRNAs that are responsible for the synthesis of proteins that for example inhibit the effects of particular antibiotics. In other aspects, solutions of PNAs can be used to disinfect objects that have been contaminated with a particular bacteria. The disinfection procedure can be designed in a specific manner thereby affecting only the bacteria that is undesired leaving other bacteria or microorganisms unaffected.
Studies using PNAs targeted to the start codon regions of xcex2-galactosidase and xcex2-lactamase genes of E. coli showed inhibitory effects both in-vitro and in-vivo. The start codon has been suggested as a viable target for antisense oligonucleotides (Sharma, H. W., Narayanan, R., BioEssays, 1995, 17, 1055-1063). In in-vitro studies the targeted PNAs showed activity and specificity. The activity was limited to the target gene mRNA and had no effect on non-targeted mRNA as seen by assays measuring protein production (Examples 1 and 2). The same study was repeated using the intact gene in comparison with two mutant genes. The activity was greatly reduced when the gene had 2 non-essential base substitutions. There was no activity observed when the mutant gene had 6 non-essential base substitutions.
Most bacteria which are resistant to a given drug also exhibit similar resistance to chemically similar drugs. Currently, many antibiotics are based on the xcex2-lactam chemical core structure of penicillin. Although other chemically diverse antibiotics, such as vancomycin, are currently available, it is only a matter of time before the emergence of bacterial strains which will be resistant to all currently available antibiotic drugs. Thus, to prevent a future world-wide epidemic of drug resistant bacterial infections, there is a never ending need for a development of antibiotic drugs with novel chemical structures (Travis, J., Science, 1994, 264, 360-362; Kingman, S., Science, 1994, 264, 363-365; and Impacts of Antibiotic-Resistant Bacteria, September, 1995, OTA-H-629, GPO stock #052-003001446-7, pages 1-8). This invention addresses this goal and many others as detailed below.
Moreover, the methods and compositions of the invention provide for control of gene expression in bacteria. When targeted to an essential gene, such as that encoding DNA Gyrase, DNA Polymerase, RNA Transcriptase, etc. (cite), the methods and compositions of the invention provide for bacteriostatic and/or bacteriocidal means for carrying out antibacterial modes of the invention.
The methods and compositions of the invention provide for an antibacterial composition comprising a peptide nucleic acid that can be used for, e.g., a disinfectant. The antibacterial compositions of the invention have bacteriostatic and/or bacteriocidal properties. In one embodiment, such antibacterial compositions comprise a peptide nucleic acid targeted to an essential bacterial nucleic acid such as, for example, a ribosomal RNA (rRNA).
The antibacterial compositions of the invention can, according the methods of the invention, be used to disinfect objects desired to be disinfected by contacting them with antibacterial compositions of the invention. Such objects to be disinfected can be composed of a number of materials (e.g., polished metals, durable plastics and the like) and include, but are not limited to, physician""s tools, including many tools used in examination rooms and more specifically, surgical tools and instruments, such as scalpels and scissors and the like; and barber""s and beauticians""s tools, such as combs, razors, and the like.
Furthermore, the compositions of the invention can be used to form antibacterial pharmaceutical compositions comprising either a PNA of the invention or such a PNA in combination with one or more other antibacterial agents. Such antibacterial pharmaceutical compositions include one or more PNAs targeted to bacterial genes or RNAs, including genes encoding DNA Gyrase, DNA Polymerase, Transcriptase, etc. In one particularly favored embodiment, the PNAs of the method targeted a gene to b-lactamase are combined with one or more b-lactam agents having antibacterial activity, e.g., penicillin, amoxocillin and the like.
The following examples are merely illustrative of the present invention and should not be considered limiting of the scope of the invention in any way. These examples and equivalents thereof will become more apparent to those skilled in the art in light of the present disclosure and the accompanying claims. The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference in their entirety.